The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death. We are studying this metabolic pathway at the level of : a) Proline oxidase - its induction and its role in apoptosis, b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule and c) Imidodipeptides and prolidase.a) Proline oxidase - its induction and its role in apoptosis. During this last year, we have focused on proline oxidase, the enzyme which catalyzes the conversion of proline to pyrroline 5-carboxylate. The enzyme is bound to mitochondrial inner membranes and donates electrons to complex II. Recently others have shown that proline oxidase (POX) is one of only 11 genes (over 7000 genes monitored by SAGE) highly induced accompanying p53-dependent induction of apoptosis in colorectal tumor cells. To investigate the role of POX, we first used RT-PCR to examine several cell types and the stimuli which will result in POX induction. In human colorectal cancer (LoVo) cells, mouse colonic epithelial cells transformed by temperature sensitive SV40 (YAMC), as well as a variety of carcinoma cell lines, we found that serum starvation as well as cytotoxic drugs could induce proline oxidase. We proposed that proline oxidase may contribute to the metabolic events accompanying apoptosis by generating reactive oxygen species (ROS). This possibility was examined in YAMC cells in which ROS was monitored with a fluorescent probe and laser cytometry. Interestingly, the addition of proline markedly increased ROS generation in a concentration- dependent manner but only accompanying adriamycin-induced apoptosis. Glutamate, on the other hand, was without effect. We are currently identifying other proline-modulated events downstream in the apoptosis pathway. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. Pyrroline 5-carboxylate (P5C), the product of proline oxidase, has been shown to have regulatory activities. Since the expression of Nitric Oxide Synthase II (NOS II) has been shown to be stimulated by amino acid metabolites, we tested the effect of P5C on NOS II expression in murine macrophages and found that P5C increased the level of NOS II protein and mRNA. The increase in NOS II mRNA was dependent on P5C concentration and duration of exposure. This effect of P5C, however, was evident only when cells were cultured under hypoxic conditions. Although NOS II expression responded to lipopolysaccharide and interferon gamma under normoxic conditions, P5C was without effect except under hypoxic conditions. We are currently defining the molecular mechanisms for this apparent transcriptional effect of P5C.c) Imidodipeptides and prolidase. Imidodipeptides containing proline or hydroxyproline originate from either tissue matrix degradation or from dietary protein. They are not substrate for generic peptidases but instead circulate to tissues where they are hydrolyzed by prolidase to release proline. Since proline may be important in apoptosis, the regulated release of proline by prolidase is of interest. We found that the level of cellular prolidase is regulated by extracellular collagen acting through integrin receptors. Thus, the hydrolysis of imidodipeptides is responsive to cellular interaction with extracellular matrix. d) ROS generation stimulated by prostate specific antigen. We are pursuing the hypothesis that prostate specific antigen (PSA), the well-known clinical marker for prostate cancer, also may be a signaling molecule. PSA is a potent stimiulator of ROS generation in LNCaP cells and may be the mediator of the effects of androgens on ROS generation. The effect of PSA on ROS is independent of androgen mechanisms. PSA affected PC-3, DU145 cells as well as LNCaP. We are investigating the involved mechanisms. - metabolism, Nitric oxide, nutrition, Oxidative stress, signaling,